The present invention relates to a method of constructing a mutant strain capable of producing amino acids in a high yield, and a method of producing L-amino acids by the fermentation with the mutant.
Methods of constructing mutant strains usable for the production of L-amino acids by the fermentation can be roughly classified into two methods. One of them comprises introducing random mutations into DNA with a chemical mutagen, and the other comprises the genetic recombination. In the latter method, a strain having an improved capacity of producing an intended substance can be developed by enhancing a gene on a metabolic pathway relating to the biosynthesis of an intended substance, or by weakening a gene of an enzyme relating to the destruction. In this connection, for enhancing an intended gene, a plasmid capable of autonomously replicating independently from the chromosome in a cell has been mainly used.
However, the method of enhancing the intended gene with a plasmid has problems. In particular, the degree of enrichment of the intended gene is variable depending on the number of copies of the plasmid itself. Therefore, for some kinds of intended genes, the copies are often too many in number and, as a result, the expression becomes excessive, the growth is seriously inhibited or the capacity of producing the intended substance is lowered. In such a case, although the degree of the enhancement of the intended gene can be lowered by using a plasmid of a small number of the copies, the variety of the plasmid is limited in many cases, and the intended control of the expression level of the intended gene is impossible.
Another problem is that since the replication of the plasmid is often unstable, the plasmid is eliminated.
For example, Japanese Patent Unexamined Published Application (hereinafter referred to as “J.P. KOKAI”) No: 61-268185 discloses a recombinant DNA comprising a DNA fragment containing a glutamate dehydrogenase (GDH)-producing gene (glutamate dehydrogenase gene) derived from a glutamate-producing coryneform bacterium, and a DNA fragment (plasmid) containing a gene necessary for the autonomous replication in the cell. It is also disclosed therein that by introducing the recombinant DNA into a cell, a GDH-enriching strain can be grown to improve the production of substances (such as amino acids and proteins) with microorganisms.
On the other hand, in Japanese Patent No. 2,520,895, the above described recombinant DNA is introduced into Corynebacterium to obtain a strain having the improved enzymatic activity, and L-glutamic acid is produced by the fermentation with the strain. However, the production and yield of L-glutamic acid were yet unsatisfactory. Thus, it is demanded to further improve the productivity of L-glutamic acid. It is reported that the demand had been attained by introducing a recombinant DNA comprising two kinds of genes, i.e. a glutamate dehydrogenase-producing gene derived from a glutamate-producing coryneform bacterium, and an isocitrate dehydrogenase (ICDH) gene, into a glutamate-producing coryneform bacterium.
Further, JP Kokai No. 6-502548 discloses an expression system and a secretion system of Corynebacterium comprising a Corynebacterium strain and a secretory cassette comprising the first functional DNA sequence for the expression in the strain, the second DNA sequence encoding for amino acids, polypeptides and/or proteins and the third DNA sequence inserted between the first DNA sequence and the second DNA sequence, wherein the third DNA sequence encodes the protein element selected from PS1 and PS2 which guarantee the secretion of the amino acids, polypeptides and/or proteins. Specifically, the secretion of polypeptides is disclosed therein and in particular, NTG mutagenesis was conducted with Corynebacterium and a mutant resistant to 4-fluoroglutamate (4FG) which is an analogue to glutamate is selected and subjected to the transformation with PCGL141. It is described therein that a strain having an enhanced expression of GDH can be obtained from the analogue resistant bacteria. It is also described therein that a mutation was observed in nucleotide sequence No.251 to No.266 of GDH promoter.